Macrophages and monocytes become effector and immunoregulatory cells when activated by biological response modifiers (BRM). We studied the molecular mechanisms of the activation process. Because of the major role of RNA in any process of cellular differentiation, we analyzed the changes in RNA metabolism occurring during the transition from resting to tumoricidal macrophage. We found a direct correlation between expression of tumoricidal activity and decrease of RNA synthesis. Molecular analysis of the species of RNA affected revealed a selective inhibition of the 28S ribosomal RNA in activated macrophages. The down-regulation of RNA synthesis was observed in macrophages activated in vitro by endotoxins, lymphokines and interferon, and in vivo by endotoxins and C. parvum. Moreover, studies of macrophages from different strains of mice showed that the cytotoxic response to activating signals and the decrease in RNA synthesis followed the same genetic distribution. We concluded that the decrease in RNA synthesis is a marker for the aquisition of cytotoxic activity by macrophages exposed to BRM. We reasoned that if the down-regulation of RNA synthesis was causally related to the activation, drugs affecting RNA synthesis would exert a positive effect in the generation of cytotoxic macrophages. Indeed we found that inhibitors of RNA synthesis, such as actinomycin D and picolinic acid can activate macrophages to a tumoricidal state. Moreover, increasing evidence has been obtained on the positive effects of inhibitors of RNA synthesis on macrophage activation by demonstrating synergy between these drugs and suboptimal doses of BRM. These findings demonstrate for the first time that the down-regulation of RNA synthesis is involved in the generation of cytotoxic macrophages. Moreover, they suggest that combined treatments of patients with BRM and inhibitors of nucleic acid synthesis could be designed in order to exploit the macrophage-mediater antitumor defenses in addition to the direct therapeutic effects of the drugs.